The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Because of its excellent metal chelating properties diethylenetriaminepentaacetic acid (DTPA) is one of the most widely used organic ligands in magnetic resonance imaging (MRI) and positron emission tomography (PET) [Aime, S., Botta, M., Fasano, M. and Terrano, E. 1998, Chem. Soc. Rev., 27, 19, Caravan, P., Ellison, J. J., McMurry, T. J. and Lauffer, R. B., 1999, Chem. Rev., 99, 2293, Woods, M., Kovacs, Z. and Sherry, A. D., 2002, J. Supramol. Chem., 2, 1]. Indeed, the first FDA approved contrast agent in clinical use is the Gd3+ DTPA chelate [Runge, V. M., 2000, J. Magn. Res. Imaging, 12, 205.]. The corresponding 111In and 68Ga chelates, in turn, are suitable for PET applications [Anderson, C. J. and Welch, M. J., 1999, Chem. Rev. 99, 2219], while Eu3+, Tb3+, Sm3+ and Dy3+ chelates can be used in applications based on dissosiation enhanced lanthanide fluorescence immunoassay (DELFIA) [PCT WO 03/076939A1]. 99mTc DTPA in turn, is suitable for single positron emission computed tomography (SPECT) [Lorberboym, M., Lampl, Y. and Sadeh, M., 2003, J. Nucl. Med. 44, 1898, Galuska, L., Leovey, A., Szucs-Farkas, Z., Garai, I., Szabo, J., Varga, J. and Nagy, E. V., 2002, Nucl. Med. Commun. 23, 1211]. Bioactive molecules labeled with 111In or 117mSn DTPA may find applications as target-specific radiopharmaceuticals [Volkert, W. A. and Hoffman, T. J., 1999, Chem. Rev. 99, 2269].
In several applications, covalent conjugation of DTPA to bioactive molecules is required. Often, isothiocyanato, N-hydroxysuccinimide or maleimide derivatives of the chelate are used in the labeling the target molecules such as oligonucleotides and oligopeptides. Several bifunctional DTPA derivatives are currently commercially available. Because in all of these cases the labeling reaction is performed in the presence of an excess of an activated label, laborious purification procedures cannot be prevented. Especially, when attachment of several label molecules is needed, purification and characterization of the desired biomolecule conjugate may be extremely difficult.
The purification problems can be avoided by performing the labeling reaction on solid phase. Hence, most of the impurities can be removed by washings when the biomolecule conjugate is still anchored to the solid support, and after release to the solution, only one chromatographic purification is needed. Several such blocks have been published. They include organic dyes [Loshe, J., Nielsen, P. E., Harrit, N. and Dahl, O., 1997, Bioconjugate Chem. 8, 503, McCafferty, D. G., Bishop, B. M., Wall, C. G., Hughes, S. G., Mecklenberg, S. L., Meyer, T. J., and Erickson, D. W., 1995, Tetrahedron, 51, 1093, WO 96/03409, Cuppoletti, A., Cho, Y., Park, J.-C., Strässler, G. and Kool, E. T. 2005, Bioconjugate Chem. 16, 528, Bethelot, T., Lain, G., Latxague, L. and Deleris, G., 2004, J. Fluorescence, 14, 671], derivatives of EDTA [Sluka, J. P., Griffin, J. H., Mack, D. P. and Dervan, P. B. 1990, J. Am. Chem. Soc, 112, 6369, Arya, R. and Gariepy, J. 1991, Bioconjugate Chem., 2, 323, Cuenoud, B. and Schepartz, A. 1991, Tetrahedron, 47, 2535, Rana, T. M., Ban, M. and Hearst, J. E., 1992, Tetrahedron Lett, 33, 4521, Song, A. I. and Rana, T. A., 1997, Bioconjugate Chem., 8, 249, Davies, J. C., Al-Jamri, L., 2002, J. peptide Sci., 8, 663, U.S. Pat. No. 5,637,759], DOTA [Bhorade, R., Weissleder, R., Nakakoshi, T., Moore, A. and Tung, C.-H., 2000, Bioconjugate Chem., 11, 301., Gallazzi, F., Wang, Y., Jia, F., Shenoy, N., Landon, L. A., Hannink, M., Lever, S. Z. and Lewis, M. R. 2003, Bioconjugate Chem., 14, 1083.] and luminescent and non-luminescent lanthanide chelates (U.S. Pat. No. 6,080,839; U.S. Pat. No. 6,949,696; Peuralahti, J., Hakala, H., Mukkala, V.-M., Hurskainen, P., Mulari, O. and Hovinen, J. 2002 Bioconjugate Chem. 13, 876.].
Although DTPA molecule is known for decades, and although reagents for solid phase oligonucleotide derivatization with DTPA has been demonstrated [U.S. Pat. No. 6,949,639], no reactants which allow its direct solid phase conjugation to oligopeptides have been synthesized. The solid phase methods published involve synthesis of oligopeptides, where one ε-amino group of lysine is selectively deprotected while the oligomer is still anchored to the resin [Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J. and Gilles, R. J. 2005, Anal. Biochem. 343, 299, Nagy, I. B., Vagra, I. and Hudecz, F, 2000, Anal. Biochem., 287, 17] Then, an activated DTPA molecule (as an anhydride or an HOBt ester) is coupled to the primary amino function, the oligopeptide is deprotected and converted to the appropriate DTPA chelate. However, this methodology has some drawbacks. First, practically only one DTPA molecule can be introduced. This may be problematic in applications were high detection sensitivity is required. Second, since one of the iminoacetic acid groups is used for conjugation, the resulting chelate is less stable than the parent DTPA molecule [Paul-Roth, C. and Raymond, K. N. 1995, Inorg. Chem. 34, 1408, Li, W. P., Ma, D. S., Higginbotham, C., Hoffman, T., Ketring, A. R., Cutler, C. S, and Jurisson, S. S. 2001, Nucl. Med. Biol. 28, 145.]. This may be a serious problem in vivo applications especially in MRI due to the high toxicity of free Gd(III) ion.
A schematic preparation of stable DTPA derivatives applicable to solid phase peptide incorporation have been proposed [U.S. Pat. No. 5,637,759], but the method of their preparation is challenging due to the carboxyl protecting strategy. There, selective deprotection of a single and specific carboxylic acid group out of six of similar reactivities is required. This problem can be avoided by changing the protecting group strategy, but the synthetic route will be considerably longer [WO 03/011115].